Delivery of poly (ethylene glycol)-modified molecules from degradable hydrogels

ABSTRACT

A degradable PEG hydrogel is described that, upon hydrolysis, releases conjugates of substantially non-peptidic polymers and biologically active molecules. For example, PEG and protein conjugates can be released in vivo from the hydrogels for therapeutic application.

This application is a divisional application of Ser. No. 09/426,289,filed Oct. 25, 1999, now U.S. Pat. No. 6,432,397, which is a divisionalapplication of Ser. No. 08/964,972 filed Nov. 5, 1997, now U.S. Pat. No.6,258,351, which is related to commonly owned copending ProvisionalApplication Ser. No. 60/030,453, filed Nov. 6, 1996, and claims thebenefit of its earlier filing date under U.S.C. 119(e).

FIELD OF THE INVENTION

This invention relates to crosslinked hydrogel networks that include thehydrophilic polymer poly(ethylene glycol).

BACKGROUND OF THE INVENTION

Chemical attachment of the hydrophilic polymer poly(ethyleneglycol)(PEG), also known as poly(ethylene oxide)(PEO), to molecules andsurfaces is of great utility in biotechnology. In its most common form,PEG is a linear polymer terminated at each end with hydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

This polymer can be represented in brief form as HO—PEG—OH where it isunderstood that the —PEG— symbol represents the following structuralunit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

In typical form, n ranges from approximately 10 to approximately 2000.

PEG is commonly used as methoxy-PEG—OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group that is subject to ready chemical modification.

CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH mPEG

PEG is also commonly used in branched forms that can be prepared byaddition of ethylene oxide to various polyols, such as glycerol,pentaerythritol and sorbitol. For example, the four-arm, branched PEGprepared from pentaerythritol is shown below;

C(CH₂—OH)₄+n C₂H₄O→C[CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH]₄

The branched PEGs can be represented in general form as R(—PEG—OH)_(n)in which R represents the central “core” molecule, such as glycerol orpentaerythritol, and n represents the number of arms.

PEG is a much used polymer having the properties of solubility in waterand in many organic solvents, lack of toxicity, and lack ofimmunogenicity. One use of PEG is to covalently attach the polymer toinsoluble molecules to make the resulting PEG-molecule “conjugate”soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem.,60, 331-336 (1995) have shown that the water-insoluble drug taxol, whencoupled to PEG, becomes water soluble.

Davis et al. U.S. Pat. No. 4,179,337 describes proteins coupled to PEGand having enhanced blood circulation lifetime because of reduced rateof kidney clearance and reduced immunogenicity. The lack of toxicity ofthe polymer and its rapid clearance from the body are advantageousfeatures for pharmaceutical applications. These applications and manyleading references are described in the book by Harris (J. M. Harris,Ed., “Biomedical and Biotechnical Applications of Polyethylene GlycolChemistry,” Plenum, N.Y., 1992).

To couple PEG to a molecule such as a protein it is necessary to use an“activated derivative” of the PEG having a functional group at theterminus suitable for reacting with some group on the surface or on theprotein (such as an amino group). Among the many useful activatedderivatives of PEG is the succinimidyl “active ester” ofcarboxymethylated PEG as disclosed by K. Iwasaki and Y. Iwashita in U.S.Pat. No. 4,670,417. This chemistry is illustrated with the active esterreacting with amino groups of a protein (the succinimidyl group isrepresented as NHS and the protein is represented as PRO—NH₂)

PEG—O—CH₂—CO₂—NHS+PRO—NH₂→PEG—O—CH₂—CO₂—NH—PRO

Succinimidyl “active esters”, such as PEG—O—CH₂—CO₂—NHS, are commonlyused forms of activated carboxylic acids, and they are prepared byreacting carboxylic acids with N-hydroxylsuccinimide.

Problems have arisen in the art. Some of the functional groups that havebeen used to activate PEG can result in toxic or otherwise undesirableresidues when used for in vivo drug delivery. Some of the linkages thathave been devised to attach functional groups to PEG can result in anundesirable immune response. Some of the functional groups do not havesufficient or otherwise appropriate selectivity for reacting withparticular groups on proteins and can tend to deactivate the proteins.

PEG hydrogels, which are water-swollen gels, have been used for woundcovering and drug delivery. PEG hydrogels are prepared by incorporatingthe soluble, hydrophilic polymer into a chemically crosslinked networkor matrix so that addition of water produces an insoluble, swollen gel.Substances useful as drugs typically are not covalently attached to thePEG hydrogel for in vivo delivery. Instead, the substances are trappedwithin the crosslinked matrix and pass through the interstices in thematrix. The insoluble matrix can remain in the body indefinitely andcontrol of the release of the drug can be somewhat imprecise.

One approach to preparation of these hydrogels is described in Embreyand Graham's U.S. Pat. No. 4,894,238, in which the ends of the linearpolymer are connected by various strong, nondegradable chemicallinkages. For example, linear PEG can be incorporated into a crosslinkednetwork by reacting with a triol and a diisocyanate to formhydrolytically-stable (“nondegradable”) urethane linkages.

A related approach for preparation of nondegradable PEG hydrogels hasbeen demonstrated by Gayet and Fortier in J. Controlled Release, 38,177-184 (1996) in which linear PEG was activated as thep-nitrophenylcarbonate and crosslinked by reaction with a protein,bovine serum albumin. The linkages formed are hydrolytically-stableurethane groups.

N. S. Chu U.S. Pat. No. 3,963,805 describes nondegradable PEG networkshave been prepared by random entanglement of PEG chains with otherpolymers formed by use of free radical initiators mixed withmultifunctional monomers. P. A. King U.S. Pat. No. 3,149,006 describesthe preparation of nondegradable PEG hydrogels by radiation-inducedcrosslinking of high molecular weight PEG.

Nagaoka et al. U.S. Pat. No. 4,424,311 describes PEG hydrogels preparedby copolymerization of PEG methacrylate with other comonomers such asmethyl methacrylate. This vinyl polymerization will produce apolyethylene backbone with PEG attached. The methyl methacrylatecomonomer is added to give the gel additional physical strength.

Sawhney, Pathak and Hubbell in Macromolecules, 26, 581 (1993) describethe preparation of block copolymers of polyglycolide or polylactide andPEG that are terminated with acrylate groups, as shown below:

CH₂═CH—CO—(O—CH₂—CO)_(n)—PEG—(O—CH₂—CO)_(n)—O—CO—CH═CH₂

In the above formula, the glycolide blocks are the —O—CH₂—CO— units;addition of a methyl group to the methylene gives a lactide block; n canbe multiples of 2. Vinyl polymerization of the acrylate groups producesan insoluble, crosslinked gel with a polyethylene backbone. Thepolylactide or polyglycolide segments of the polymer backbone, beingester groups, are susceptible to slow hydrolytic breakdown, with theresult that the crosslinked gel undergoes slow degradation anddissolution.

Substantial non-PEG elements are introduced into the hydrogel. Non-PEGelements tend to introduce complexity into the hydrogel and degradationand dissolution of the matrix can result in undesirable or toxiccomponents being released into the blood stream when the hydrogels areused in vivo for drug delivery.

It would be desirable to provide alternative PEG hydrogels that aresuitable for drug delivery and that have unique properties that couldenhance drug delivery systems.

SUMMARY OF THE INVENTION

The invention provides chemically crosslinked PEG hydrogels forcontrolled release of conjugates of PEG and various molecules,including, for example, conjugates of PEG and enzymes, polypeptides,drugs, nucleosides, phospholipids, and other bioactive substances. Theinvention also provides methods for preparing the hydrogels.

The hydrogels of the invention are formed by reaction of activederivatives of poly(ethylene glycol) with amine groups on the bioactivesubstance or other molecule and with amine groups on other poly(ethyleneglycol) molecules or related similar nonpeptidic polymers that typicallydo not contain hydrolytically unstable linkages. The poly(ethyleneglycol) molecules that contain weak linkages in their backbones permithydrolytic degradation of the crosslinks in the polymer matrix andrelease of the bioactive substance with the other poly(ethylene glycol)or related nonpeptidic polymer attached. Degradation of the gel in vivoreleases PEG/molecule conjugates into the blood stream and producessubstantially nontoxic polymer fragments that typically are cleared fromthe body. Variation of the atoms near the hydrolytically unstablelinkages can provide precise control of hydrolytic breakdown rate andrelease of the conjugate.

Examples of hydrolytically unstable linkages in the PEG polymer backboneinclude carboxylate ester, phosphate ester, acetals, imines,orthoesters, peptides, anhydrides, ketals, and oligonucleotides. Theseweak links are formed by reaction of two PEGs having different terminalgroups as illustrated below:

—PEG—Z+Y—PEG—→—PEG—W—PEG—

In the above illustration, —W— represents the hydrolytically unstableweak link. Z— and Y— represent groups located at the terminus of the PEGmolecule that are capable of reacting with each other to form weak links—W—. Examples of pairs of Z and Y groups that react to formhydrolytically unstable linkages W include pairs selected from the groupconsisting of alcohol, and carboxylic acid reacting to form carboxylateesters, amine and aldehyde reacting to form imines, hydrazide andaldehyde reacting to form hydrozones, alcohol and phosphate reacting toform phosphate ester, aldehyde and alcohol reacting to form acetals,alcohols and formate reacting to form orthoesters, peptides formed bythe reaction of PEG amine with PEG-peptide terminated with carboxyl toform a new peptide linkage, peptides formed by the reaction of PEGcarboxylic acid with PEG-peptide terminated with amine to form a newpeptide linkage, and oligonucleotides formed by reaction of PEGphosphoramidite with an 5′-hydroxyl-terminated PEG oligonucleotide.

For example, the following pairs of Z and Y groups can be used to formsome of the W groups described above:

—PEG—CO₂H+HO—PEG—→—PEG—CO₂—PEG— ester

—PEG—OPO₃H₂+HO—PEG—→—PEG—OPO₃(H)—PEG— phosphate ester

—PEG—CHO+(HO—PEG)₂—→—PEG—CH(O—PEG)₂— acetal

—PEG—CHO+NH₂—PEG—→—PEG—CH═N—PEG— imine

The PEG hydrogels gels are prepared by mixing three ingredients: (1) aPEG with hydrolytically unstable linkages W in the backbone and withreactive groups X at the ends of the chain, (2) a branched PEG orrelated nonpeptidic polymer with reactive groups Q at the ends of thechain, and (3) a bioactive molecule or other molecule containingreactive groups Q. Reactive groups X are selected from the groupconsisting of succinimidyl (NHS), as in —O—(CH₂)_(n)—CO₂—NHS or—O—CO₂—NHS, and related activating groups, including sulfosuccinimidyl,benzotriazole, and p-nitophenyl. Reactive groups Q typically are amine,—NH₂.

A crosslinked network is produced that is held together byhydrolytically unstable groups W and groups T, which are hydrolyticallystable. Hydrolysis of the unstable groups W releases the bioactive orother molecule with PEG or a related polymer attached, usually by acovalent linkage, which is hydrolytically stable.

The degree of branching of the polymers can be varied in the hydrogelsof this invention to control the physical strength and compressibilityof the gels. In general, the greater the degree of branching and theshorter the branches, the greater the strength of the gels, the smallerthe pores, and the lower the water content. Strength in this context isdefined as resistance to compression or stretching.

The rate of release of molecules trapped within the hydrogel matrix iscontrolled by controlling the hydrolytic breakdown rate of the gel. Thehydrolytic breakdown rate of the gel can be adjusted by controlling thedegree of bonding of the PEGs that form the hydrogel matrix. A multiarmPEG having 10 branches or arms will break down and release drugmolecules more slowly than a 3 arm PEG.

The following PEG has been made with two hydrolytically unstable esterlinkages in its backbone:

NHS—O₂C—CH₂—O—PEG—O—CH₂—CO₂—PEG—O₂C—CH₂—O—PEG—O—CH₂—CO₂—NHS

The above PEG is activated at each terminus with anN-hydroxylsuccinimide moiety (NHS) in which the active succinimidylester moiety is NHS—CO₂— and is reactive with amino groups. Acrosslinked network is produced that is held together by stable amidelinkages and by hydrolytically unstable ester linkages when the abovemolecule is coupled with a multiarm PEG amine and with, for example, aprotein that contains additional amino groups. The stable amide linkagesare formed from reaction of the active NHS ester with amine.

The above example illustrates some of the advantageous features of theinvention. First, the crosslinked network degrades or breaks downbecause of hydrolysis of the hydrolytically unstable ester linkages (W)in the PEG backbone. Second, when the gel breaks down, it releases PEGand protein conjugates, potentially useful for therapeutic application.Third, subtle variation of the ester linkage provides control over thehydrolytic breakdown rate.

In the above example the ester linkage has the following structure:

—PEG—O—CH₂—CO₂—PEG—

This ester group will hydrolyze with a half life of 4 days at pH 7 and37° C. However, if an ester with the following structure is used, thenthe half life of hydrolytic degradation of the ester linkages is 43 daysat pH 7 and 37° C.

—PEG—O—(CH₂)_(n)—CO₂—PEG— n=2

Thus, by controlling the identity of the atoms adjacent to the esterlinkage it is possible to vary the hydrolytic breakdown rate of the gel.Hence, it is possible to control the rate of release of PEG and proteinconjugates bound within the matrix. In general, increasing the n value,which is the number of methylene groups in the above structure,decreases the hydrolysis rate.

Thus, the invention provides, among other things, degradable PEGhydrogels having hydrolytically unstable linkages in which the rate ofhydrolysis of the unstable linkages can be controlled for release intothe blood stream of conjugates of PEG or related nonpeptidic polymersand proteins or other molecules having some therapeutic effect.

The foregoing and other objects of the invention, and the manner inwhich the same are accomplished, will be more readily apparent uponconsideration of the following detailed description of the inventiontaken in conjuction with the accompanying drawing, which illustrates anexemplary embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a release profile from a PEG hydrogel prepared in accordancewith the invention of a model protein (FITC-BSA) covalently linked toPEG.

DETAILED DESCRIPTION

Hydrogels made from the crosslinked PEG polymeric structures of theinvention can be used in drug delivery systems and for wound dressings.Wound dressings could be used internally to provide dressings thatdegrade within the body over time. The hydrogels of the invention couldbe usefully applied in drug delivery systems to burns to apply polymerconjugated therapeutic agents to burns. Drug delivery systems can beprepared in which the rate of hydrolysis of the hydrogel is controlledto provide controlled release of drug components.

By “drug” is meant any substance intended for the diagnosis, cure,mitigation, treatment, or prevention of disease in humans and otheranimals, or to otherwise enhance physical or mental well being. Theinvention could be used for delivery of biologically active substancesgenerally that have some activity or function in a living organism or ina substance taken from a living organism.

The terms “group,” “functional group,” “moiety,” “active moiety,”“reactive site,” and “radical” are all somewhat synonymous in thechemical arts and are used in the art and herein to refer to distinct,definable portions or units of a molecule and to units that perform somefunction or activity and are reactive with other molecules or portionsof molecules.

The term “linkage” is used to refer to groups that normally are formedas the result of a chemical reaction and typically are covalentlinkages. Hydrolytically stable linkages means that the linkages arestable in water and do not react with water at useful pHs for anextended period of time, potentially indefinitely. Hydrolyticallyunstable linkages are those that react with water, typically causingdegradation of a hydrogel and release of substances trapped within thematrix. The linkage is said to be subject to hydrolysis and to behydrolyzable. The time it takes to degrade the crosslinked polymericstructure is referred to as the rate of hydrolysis and is usuallymeasured in terms of its half life.

The skilled artisan should recognize that when reference is made to a Zmoiety reacting with a Y moiety, that additional reagents or steps maybe employed according to commonly accepted chemical procedures andstandards to achieve the desired linkage W as the case may be. There aremany possible routes, too numerous to mention here, that could be takenand that should be readily apparent to the skilled artisan. For example,one of skill in the art can be expected to understand that when analcohol and a carboxylic acid are reacted, the acid typically isconverted to another form, the acid chloride, prior to reaction withalcohol. Several examples are demonstrated in the Examples below.

It should also be recognized that related branched nonpeptidic polymersthat do not have hydrolytically unstable linkages can be used instead ofthe branched PEG polymer as an ingredient in the preparation of thehydrogels of the invention. These other branched polymers includepoly(vinyl alcohol) (“PVA”); other poly(alkylene oxides) such aspoly(propylene glycol) (“PPG”) and the like; and poly(oxyethylatedpolyols) such as poly(oxyethylated glycerol), poly(oxyethylatedsorbitol), and poly(oxyethylated glucose), and the like. The polymerscan be homopolymers or random or block copolymers and terpolymers basedon the monomers of the above polymers, straight chain or branched, orsubstituted or unsubstituted similar to mPEG and other capped,monofunctional PEGs having a single active site available for attachmentto a linker.

Specific examples of suitable additional polymers includepoly(oxazoline), poly(acryloylmorpholine) (“PAcM”) as described inpublished Italian Patent Application MI-92-A-0002616 filed Nov. 17,1992, and poly(vinylpyrrolidone) (“PVP”). PVP and poly(oxazoline) arewell known polymers in the art and their preparation and use in thesyntheses described with branched PEG should be readily apparent to theskilled artisan.

The following examples illustrate preparation of PEGs havinghydrolytically unstable linkages in the polymer backbone and their usein preparing degradable hydrogels for the release of PEG and biomoleculeconjugates. PEGs having hydrolytically unstable linkages and theirpreparation are also described in a copending patent application U.S.Ser. No. 08/928,049, entitled Degradable Poly(ethylene glycol) HydrogelsWith Controlled Half-life and Precursors Therefor, which was filed onSep. 12, 1997 and claims priority from Provisional Application Ser. No.60/026,066, which was filed on Sep. 13, 1996, the contents of whichrelating to the preparation of PEGs having hydrolytically unstablelinkages in the polymer backbone are incorporated by reference in theirentirety.

EXAMPLES Example 1 Synthesis of PEG Derivatives Having HydrolyticallyUnstable Backbone Linkages and Terminal NHS Active Carbonates(NHS—OOCO—PEG—W—PEG—OCOO—NHS)

In a 100 ml round-bottom flask, benzyloxy-PEG carboxymethyl acid 3400(3.4 g, 1 mmol, Shearwater Polymers, Huntsville, Ala.) in toluene wasazeotropically distilled for two hours and then cooled to roomtemperature. A solution of thionyl chloride (2M, 4 ml, 8 mmole, Aldrich)in methylene chloride was injected and the mixture was stirred under N₂overnight. The solvent was condensed by rotary evaporation and the syrupwas dried in vacuo for about four hours over P₂O₅ powder. To the residuewas added anhydrous methylene chloride (5 ml) and azeotropically driedbenzyloxy-PEG 3400 (2.55 g, 0.75 mmol) in toluene (20 ml). After thebenzyloxy-PEG acyl chloride was dissolved, freshly distilledtriethylamine (0.6 ml) was added. The mixture was stirred overnight, thetriethylamine salt filtered off, and the product collected byprecipitation with ethyl ether. It was further purified by dissolving inwater and extracting with methylene chloride. The organic phase wasdried over anhydrous sodium sulfate, condensed under vacuum, andprecipitated into ethyl ether. The precipitate was dried in vacuo. HPLC(GPC) of the product showed that 100% of benzyloxy-PEG had beenconverted into the PEG ester and about 15 wt % benzyloxy-PEG acidremained.

The mixture was chromatographically purified on an ion-exchange column(DEAE sepharose fast flow, Pharmacia) to remove the benzyloxy-PEG acid.100% pure α-benzyloxy-ω-benzyloxy PEG ester 6800 (2 g, 0.59 mmole endgroup) in 1,4-dioxane (20 ml) was hydrogenolyzed with H₂ (2 atmpressure) and Pd/C (1 g, 10% Pd) overnight. The catalyst was removed byfiltration and the product precipitated into ethyl after most of thesolvent was removed on a rotary evaporator. α-hydroxy-ω-hydroxy PEGester 6800 was collected by filtration and dried in vacuo. Yield: 1.5gram (75%)

α-hydroxy-ω-hydroxy PEG ester 6800 (1.5 g, 0.44 mmole end group) wasazeotropically dried with 100 ml of acetronitrile and cooled to roomtemperature. To this solution was added disuccimidyl carbonate (DSC)(0.88 mmole, Fluka) and pyridine (0.1 ml), and the solution was stirredat room temperature overnight. The solvent was removed under vacuum andthe syrup was dried in vacuo. The product was dissolved in 35 ml of drymethylene chloride, the insoluble solid was removed by filtration, andthe filtrate washed with pH 4.5 sodium chloride saturated acetatebuffer. The organic phase was dried over anhydrous sodium sulfate,condensed under vacuum, and precipitated into ethyl ether. Theprecipitate was dried over P₂O₅ in vacuo. Yield: 1.4 g (93%). NMR(DMSO-d₆): (1) product from benzyloxy-PEG propionic acid: δ 3.5 (br m,PEG), 2.55 (t, —OCH₂ CH₂COOPEG—), 4.13 (t, —PEG—COOCH ₂CH₂O—), 4.45 (t,—PEGOCH₂CH ₂OCO—NHS), 2.80 [s, NHS, 4H]; (2) product from benzyloxy-PEGcarboxymethyl acid: δ 3.5 (br m, PEG), 4.14 (s, —OCH ₂COOPEG—), 4.18 (t,—OCH₂COOCH ₂CH₂—), 4.45 (t, —PEGO—CH₂CH ₂OCONHS), 2.81 [s, NHS, 4H].

Example 2 Synthesis of PEG Derivatives Having Hydrolytically UnstableBackbone Linkages and Terminal NHS Active Esters(NHS—OOC—(CH₂)_(n)—O—PEG—O—(CH₂)_(n)—CO₂—PEG—O₂C—(CH₂)_(n)—O—PEG—O—(CH₂)_(n)—COONHS)

In a 100 ml round-bottom flask, difunctional PEG 2000 (2 g, 1 mmol,Shearwater Polymers) and difunctional PEG acid 2000 (4 g, 2 mmole,Shearwater Polymers) were azeotropically distilled with 70 ml of tolueneunder N₂. After two hours, the solution was cooled to room temperatureand stannous 2-ethylhexanoate (200 mg, Sigma Chemical) was added. Thesolution was then refluxed under N₂ for 24 hours. The solvent was thencondensed under vacuum and the syrup precipitated into 100 ml of ether.The product was collected by filtration, dried under vacuum, anddissolved in a sodium acetate buffer solution at pH 5.0. The slightlymilky solution was centrifuged and the upper clear solution wasextracted three times with methylene chloride. The organic phase wasdried over anhydrous sodium sulfate, filtered, condensed under vacuum,and precipitated into ether. The product was collected by filtration anddried under vacuum. HPLC: 70% product, 15% di-acid reactant and 15%monoacid. The mixture was further purified by ion exchangechromatography and gel permeation chromatography. Yield 3 g (50%). ¹HNMR (DMSO-D₆): (1) product from PEG carboxymethyl acid: δ 3.5 (br m,PEG), 4.15 (s, —OCH ₂COOCH₂—), 4.18 (t, —OCH₂COOCH ₂CH₂—), 3.98 (s,—PEG—OCH₂COOH); (2) product from PEG propionic acid: δ 3.5 (br m, PEG),2.55 (t, —PEGOCH₂CH ₂COOCH₂—), 4.13 (t, —OCH₂CH₂COOCH ₂CH₂—), 2.43 (t,—PEGOCH₂CH ₂COOH).

In a round-bottom flask, the difunctional acid having weak linkages(obtained from previous step) 3 g. approx. 1 mmole end group) andN-hydroxysuccinimide (NHS) (126 mg, 1.05 mmole) were dissolved in 50 mlof dry methylene chloride. To this solution was addeddicyclohexylcarbodiimide (240 mg, 1.15 mmole) in 5 ml dry methylenechloride. The mixture was stirred under N₂ overnight. The solvent wascondensed and the syrup was redissolved in 15 ml of anhydrous toluene.The insoluble salt was removed by filtration and the filtrate wasprecipitated into 200 ml of dry ethyl ether. The precipitate wascollected by filtration and dried in vacuo. Yield 2.7 g (90%). ¹H NMR(DMSO-d₆): δ 3.5 (br m, PEG), 2.8 (s, NHS, 4H), 4.6 (s, —PEG—O—CH₂—COONHS) or 2.85 (t, —PEG—O—CH₂CH ₂—COONHS).

Example 3 Hydrolysis Kinetics of the Ester Linkages in the Middle of thePEG Derivatives

To precisely measure the hydrolysis kinetics of the ester linkages,water-soluble, non-crosslinked mPEG-O—(CH₂)_(n)—COO—PEGm was synthesizedas in Example 2. Hydrolysis was carried out in buffer solutions (0.1 M)at different pHs and temperatures, and followed by HPLC-GPC(Ultrahydrogel® 250, Waters). The half-lives of the ester bonds arelisted in Table 1.

TABLE 1 Hydrolysis half lives (days, ±10%) of the ester ofmPEG-O—(CH₂)_(n)—COO-PEGm in 0.1 M phosphate buffer. PA ester linkage CMester linkage pKa of the 4.45 ± 0.1 3.67 ± 0.05 acid pH 5.5 7.0 8.1 5.57.0 8.1 Room Temp. >500 250 37 >150 30 5 (22-23° C.) 37° C. 43 4 50° C.15 1.5

Example 4 Preparation of a Hydrolytically Unstable PEG Hydrogel fromBranched PEG Amine, Model Protein (FITC-BSA) and PEG Derivatives HavingHydrolytically-unstable Backbone Linkages and Terminal NHS ActiveCarbonates (NHS—OOCO—PEG—W—PEG—OCOONHS)

In a test tube, 100 mg (14.7 μmole) of difunctional PEG active carbonate6800 (NHS—OOCO—PEG—W—PEG—OCOONHS, prepared in Example 1) was dissolvedin 0.75 ml of buffer (0.1M phosphate, pH 7). To the solution were added0.15 ml of 8-arm-PEG-amine 10000 (250 mg/ml) and 0.1 ml of FITC-BSA (10mg/ml). After rapid shaking, it was allowed to sit and a gel formed in afew minutes. A suitable buffer pH range was found to be 5.5 to 8.

Example 5 Preparation of a Hydrolytically Unstable PEG Hydrogel fromBranched PEG Amine, Model Protein, and PEG Derivatives HavingHydrolytically Unstable Backbone Linkages and Terminal NHS Active Esters(NHS—OOC—(CH₂)_(n)—O—PEG—O—(CH₂)_(n)—CO₂—PEG—O₂C—(CH₂)_(n)—O—PEG—(CH₂)_(n)—COONHS)

100 mg (approx. 16.6 μmole) difunctional PEG active ester(NHS—OOC—(CH₂)_(n)—O—PEG—O—(CH₂)_(n)—CO₂—PEG—O₂C—(CH₂)_(n)—O—PEG—O—(CH₂)_(n)—COONHS,prepared in Example 2) was dissolved in 0.75 ml of buffer (0.1Mphosphate, pH 7). To the solution were added 0.166 ml of 8-arm-PEG-amine10000 (250 mg/ml) and 0.1 ml of FITC-BSA (10 mg/ml). After rapidshaking, it was allowed to sit and a gel formed in a few minutes. Asuitable buffer pH range was found to be 5.5 to 8.

Example 6 Studies of Release of Model Proteins from HydrolyticallyDegradable Hydrogels

All protein-loaded hydrogel disks were weighed and their diametersmeasured before release studies. Then each gel disk was immersed, attime t=0, in phosphate buffer (0.1 M, pH 7.0). The amount of the bufferwas more than 50 times that of the wet gel weight. The solution wasmaintained at 37° C., and gently shaken. At a predetermined time, asmall amount of buffer solution was removed for protein concentrationdetermination and then put back after measurement. The proteinconcentration was determined by UV measurement at 495 nm. FIG. 1 showssome release profiles of PEG-FITC-BSA from the hydrogels in unitsplotted against time in days of the fraction of moles at time t dividedby the moles at infinity, which is defined as the completion ofdegradation of the hydrogel.

The invention has been described in particular exemplified embodiments.However, the foregoing description is not intended to limit theinvention to the exemplified embodiments, and the skilled artisan shouldrecognize that variations can be made within the scope of the inventionas described in the foregoing specification. The invention includes allalternatives, modifications, and equivalents that may be included withinthe true spirit and scope of the invention as defined by the appendedclaims.

That which is claimed is:
 1. A method for applying a therapeutic agentto wounds and scars, comprising applying a crosslinked polymericstructure to the wound or scar, the crosslinked polymeric structurecomprising a poly(ethylene glycol) molecule covalently attached to thetherapeutic agent and having at least one hydrolytically unstablelinkage in its backbone, and a branched, substantially non-peptidicpolymer crosslinked to the poly(ethylene glycol) molecule, wherein thehydrolytically unstable linkage in the backbone of the poly(ethyleneglycol) molecule is capable of degrading in aqueous solution to releasea conjugate of the therapeutic agent and poly(ethylene glycol).
 2. Themethod of claim 1, wherein the branched, substantially non-peptidicpolymer is a polymeric amine having a polymer backbone selected from thegroup consisting of poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), poly(oxazoline), poly(vinylpyrrohidone),poly(acryloylmorphohine), and copolymers or terpolymers thereof.
 3. Themethod of claim 1, wherein the branched, substantially non-peptidicpolymer is selected from the group consisting of poly(alkylene oxides),poly(oxyethylated polyols), poly(olefinic alcohols), poly(oxaoline),poly(vinylpyrrolidone), poly(acryloylmorpholine), and copolymers orterpolymers thereof.
 4. The method of claim 1, wherein the branched,substantially non-peptidic polymer does not have hydrolytically unstablelinkages in its backbone.
 5. The method of claim 1, wherein thebranched, substantially non-peptidic polymer is poly(ethylene glycol).6. The method of claim 1, wherein the at least one hydrolyticallyunstable linkage is selected from the group consisting of carboxylateester, phosphate ester, orthoester, anhydride, imine, acetal, ketal,oligonucleotide, and peptide.
 7. The method of claim 1, wherein thebranched, substantially non-peptidic polymer has the structureR(CH₂—O-poly-NH₃)_(p), prior to crosslinking, wherein R is a centralbranching group, p is 3 to 10, and poly is a polymer selected from thegroup consisting of poly(alkylene oxides), poly(oxyethylaled polyols),poly(olefinic alcohols), poly(oxazoline), poly(vinylpyrrolidone)poly(acryloylmorpholine), and copolymers or terpolymers thereof.
 8. Themethod of claim 7, wherein poly is poly(ethylene glycol).
 9. The methodof claim 7, wherein R is selected from the group consisting of glycerol,glycerol oligomers, pentaerythritol, sorbitol, trimethyolpropane, anddi(trimethylolpropane).
 10. The method of claim 1, wherein thetherapeutic agent is selected from the group consisting of enzymes,polypeptides, drugs, nucleosides, and phospholipids.
 11. The method ofclaim 1, wherein the therapeutic agent comprises at least one aminegroup.
 12. The method of claim 1, wherein the covalent linkage betweenthe therapeutic agent and the poly(ethylene glycol) molecule having atleast one hydrolytically unstable linkage in its backbone is ahydrolytically stable linkage.
 13. The method of claim 12, wherein thehydrolytically stable linkage is selected from the group consisting ofamide, urethane, amine, ether, thioether, and urea.
 14. The method ofclaim 1, wherein the poly(ethylene glycol) molecule has at least twohydrolytically unstable linkages in its backbone.
 15. The method ofclaim 1, wherein the poly(ethylene glycol) molecule has the formulaX—PEG—W—PEG—T—D prior to crosslinking, wherein X is a terminal reactivegroup, W is a hydrolytically unstable linkage, T is a hydrolyticallystable linkage, and D is the therapeutic agent.
 16. The method of claim15, wherein X is selected from the group consisting of succinimidylester, sulfosuccinimidyl, benzotriazole, and p-nitrophenyl.
 17. Themethod of claim 15, wherein X is —O—(CH₂)_(n)—CO₂—NHS or —O—CO₂NHS,wherein n is 1-10.
 18. The method of claim 15, wherein W is selectedfrom the group consisting of carboxylate ester, phosphate ester,orthoester, anhydride, imine, acetal, ketal, oligonucleotide, andpeptide.
 19. The method of claim 15, wherein W is —O—(CHR′)_(r)—CO₂—,wherein r is 1-10, and R′ is hydrogen or alkyl.
 20. The method of claim1, wherein the hydrolytically unstable linkage is an ester linkage—O—(CHR′)_(r)—CO₂—, wherein r is 1-10, and R′ is hydrogen or alkyl. 21.A method for applying a therapeutic agent to wounds and scars,comprising applying a crosslinked polymeric structure to the wound orscar, the crosslinked polymeric structure comprising a poly(ethyleneglycol) molecule covalently attached to the therapeutic agent through ahydrolytically stable linkage and having at least one hydrolyticallyunstable linkage in its backbone, and a branched poly(ethylene glycol)amine crosslinked to the poly(ethylene glycol) molecule, wherein thehydrolytically unstable linkage in the backbone of the poly(ethyleneglycol) molecule is capable of degrading in aqueous solution to releasea conjugate of the therapeutic agent and poly(ethylene glycol).
 22. Themethod of claim 21, wherein the hydrolytically unstable linkage isselected from the group consisting of carboxylate ester, phosphateester, orthoester, anhydride, imine, acetal, ketal, oligonucleotide, andpeptide.
 23. The method of claim 21, wherein the hydrolytically unstablelinkage is an ester linkage —O—(CHR′)_(r)—CO₂—, wherein r is 1-10, andR′ is hydrogen or alkyl.
 24. The method of claim 21, wherein thehydrolytically stable linkage is selected from the group consisting ofamide, urethane, amine, ether, thioether, and urea.